Capillary electrophoresis ("CE") and associated capillary scale technologies provide very important analytic techniques for separation and quantitation of large biomolecules. Although such techniques are useful in separating and detecting small ions, ion chromatography has been a more dominant technique. The more successful ion chromatography detection techniques have recently been found to be applicable to capillary electrophoresis. One result has been so-called suppressed conductometric capillary electrophoresis separation systems ("SuCCESS"). SuCCESS technology can produce low .mu.g/L limits of detection for a variety of small ions in a robust manner without special efforts towards pre-concentration. (See U.S. Pat. Nos. 5,358,612 and 5,433,838 to Dasgupta and Bao.)
However, capillary electrophoresis is most commonly carried out using UV-Vis absorptiometric detection, as shown in FIG. 1. (See for example, "Capillary Electrophoresis" by S. F. Y. Li, Elsevier, N.Y. 1992. As shown in FIG. 1, a CE analysis system 10 includes a separation capillary 20 whose distal tip 30 initially is in fluid communication with a solution 40 containing analyte samples A, and typically also containing other substances X. Solution 40 is retained in a source vessel 50 and is electrically coupled by an electrode 55 by a wire 57 to a power source 60 that is at a high voltage ("HV") potential V1, typically many kilovolts. A second or ground electrode 155 is often disposed in a final destination vessel 160. As shown in FIG. 1, capillary 20 passes through a UV-visible absorption detector 90 before reaching final destination vessel 160.
Coupling HV power supply 60 to capillary 20 as shown in FIG. 1 results in a left-to-right direction migration of analyte A within the capillary, as indicated by the rightward-pointing arrows. Such migration can commence within seconds of energizing power supply 60. Power supply 60 may then be turned-off, after which tip 30 of capillary 20 may be relocated into a second vessel 70 containing running electrolyte 80. Power supply 60 is coupled to solution 80 via an electrode 55, which may be identical to (or indeed the same as) electrode 55 described in conjunction with vessel 50. Power source 60 may then be re-energized, which continues the downstream migration of the sample analyte. This type of electric field induced analyte injection is termed electromigrative or electrokinetic injection ("EI").
The distal end of capillary 140 is in fluid communication with electrolyte 150 contained in a terminating electrolyte reservoir 150. Preferably electrolyte 150 is the same as running electrolyte 80, and in the embodiment shown is at ground potential.
As noted, during EI, HV is applied with the background electrolyte (BGE)-filled capillary dipped in a sample vial. In a typical situation, electroosmotic and electrophoretic movements act together to introduce the desired class of analyte ion(s) into the capillary. In general, when the electroosmotic mobility (.mu..sub.eo) is small relative to the electrophoretic mobility (.mu..sub.eo), conditions are most favorable for electromigrative preconcentration. Under these conditions a significant amount of analyte can be introduced without the concomitant introduction of a significant liquid volume. EI has been widely used for the trace analysis. This is especially valuable with UV-Vis detection because on-column UV-Vis absorption detectors, e.g., detector 90, typically used in CE provide relatively poor concentration detection limits. In the determination of small ions, where indirect detection is typically used, the situation is even less favorable than with direct detection.
In EI, when the sample ionic strength is very low, best results are obtained if a low mobility ion is deliberately added to the sample at a concentration that is high relative to the total concentration of the analyte. By "low mobility" what is meant is an ion having mobility lower than any of the analyte ions of interest. In such case, the added ion behaves like a terminating electrolyte and electromigrative preconcentration closely resembles isotachophoresis. In some situations, a high mobility ion is the analyte of interest and low mobility ions are already present in abundance, such as in the determination of residual sulfate in sulfonate dyes. There is no need to add any terminating electrolytes in such cases. In cases where the analyte of interest is present at a low concentration in a sample that has a significant ionic strength, it is impractical to add sufficient terminating electrolyte to make the latter the dominant current carrier.
Unfortunately, even under identical sample analyte concentrations and instrumental settings, the amount of an analyte introduced into an EI system is a strong function of sample conductance. This relationship occurs because conductance affects the rate of electroosmotic introduction. Less directly conductance also affects the rate of the electrophoretic movement through a change in the field strength experienced by the sample. Further, EI is dependent on the mobility of the analyte itself, creating a bias in favor of the high mobility ions. By "bias" it is meant that the injected sample differs from the original sample. The difference occurs because there is a relative deficit of slower moving ions, and a relative excess of faster moving ions in the aliquot injected portion as contrasted to what was originally present in the sample. Although researchers such as Lee and Yeung, Anal. Chem. 1992, 64, 1226-1231, have advanced a simple approach to improve precision of the results obtained in EI through monitoring system current, the Lee-Yeung technique does little to solve the problem caused by biased injection.
Other attempts have been made in the prior art to address the above-noted bias dependency of sample conductivity. For example, the use of two separate internal standards that bracket the entire range of analyte mobilities of interest has been suggested, as has been the standard addition of every analyte of interest. These approaches are unsatisfactory and indeed can be tedious.
Finally, the prior art has tended to overlook the fundamental fact that the EI of sample ions into a capillary is ultimately dependent upon the local electrical field. Any changes in the geometry and or the physical distance between the HV electrode and the capillary tip can profoundly affect EI. Unfortunately, it has been difficult in the prior art to reliably produce a truly symmetrical electric field.
In the prior art, the total amount of analytes in the sample aliquot from which analyte ions are EI-introduced into the capillary is very large relative to the amount of analytes actually introduced. If one could perform EI from a truly small sample volume for a long enough period, it would be possible, in principle, to introduce virtually all the analyte ions of interest into the capillary in an exhaustive manner. The sample volume would not become deionized or become non-conductive in the process. Deionization or conductivity loss would not occur because electro-generated H.sup.+ or OH.sup.-, and the appropriate counter-ion already present in the sample, and those migrating against the EOF into the sample from the capillary, would maintain the sample conductive. Indeed, if EI could be carried out long enough, significant amounts of H.sup.+ or OH.sup.- would be introduced. Unfortunately exhaustive electromigration cannot be effectively practiced in the prior art.
Thus, there is a need for an easily produced microreservoir, preferably having a sub-.mu.L liquid capacity, that can be used in exhaustive electromigration and electrophoresis. Preferably such microreservoir should permit the entire sample within to be readily subjected to a symmetrical electric field. Further, the microreservoir should permit the entry tip of a separation capillary to be disposed at the symmetrical center of the sample. A system incorporating these features and methodology would advantageously reduce the effects of conductivity in electromigration injection capillary electrophoresis.
The present invention provides such a method and apparatus.